Method of blocking cytotoxic activity in patients with amyotrophic lateral sclerosis using protein V

ABSTRACT

The present invention is directed to a method of blocking the cytotoxic activity of FcγRIII receptor-positive immune cells in a patient with amyotrophic lateral sclerosis using protein V specific for IgG1 and/or IgG3. In one aspect of the invention, protein V blocks the binding of IgG1 and/or IgG3 to the FcγRIII receptor, which inactivates the receptor, and destroys cellular forms containing the receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of PCT International ApplicationNo. PCT/DE 97/02883 under 35 U.S.C. §371, filed on Dec. 10, 1997, thewhole of which is hereby incorporated by reference herein.

This application is a division of Ser. 09/367,011 filed Sep. 7, 1999which is a 371 of PCT/DE97/02883 Dec. 10, 1997.

FIELD OF THE INVENTION

The invention relates to the use of substances having a well-directed,i.e. selective, effect on a certain receptor family (FcγR) or on thoseimmune system cells with defined surface characteristics which expresssaid receptor and whose presence—in examinations performed by theapplicant—has been found to be specific, or its number specificallyincreased, in the case of amyotrophic lateral sclerosis.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (in the following referred to as ALS, itsabbreviation) is a neurodegenerative disease of the human motoneuronsystem which usually takes a lethal course within 3 to 5 years, whosecauses have not been determined etiologically and for which there is no,or no significant, therapy as yet. The progressive decay of nerve cellsof the first and second motor neurons are the cause of an increasingparalysis of the voluntary muscles, eventually leading to a totalwalking inability and the increasing paralysis of the respiratorymusculature. It has largely been proven that cellular and humoral(antibody-mediated) immunological processes play an important role, ifyet unexplained in the individual case, in the pathogenesis of ALS.Worldwide, the prevalence of this disease is 4 in 100,000 and itsincidence is 1 in 100,000 inhabitants.

Numerous examination results seem to imply that immunological mechanismsare at play in the pathogenesis of amyotrophic lateral sclerosis. Thefollowing findings substantiate this assumption: Cytotoxic serumactivity of ALS patients in neuronal cell cultures; serum immunoglobulinG (IgG) toxicity against spinal and cortical neurons as well asvoltage-dependent calcium channel proteins; cytotoxicity of thecerebrospinal fluid of ALS patients against glutamate receptors; changesof the serum concentration of IgG isotypes; immune response ofperipheral blood lymphocytes of ALS patients to isolated cell membranes;detection of invasive immune system cells in the motoneuron system ofALS patients Here, these cells seem to be involved in the motoneurondamaging mechanisms (the quotations of the individual above data can befound in: Westarp, M. E. et al., Neurosci. Lett. 173, 124-126, 1994).

Although this data seems to imply a probable involvement of the cellularimmune system in ALS, it has been impossible so far to detect definitequantitative or qualitative changes in the cellular immune system in thecase of ALS. In particular, no ALS specific cell types have so far beenfound in the blood of ALS patients which would be identifiable by theirmolecular cell surface characteristics and clearly differ from immunecells of healthy experimentees or present in other neurologicaldiseases. Nevertheless, countless therapies involving medication with arelatively broad effectivity as well as an undirected action wereattempted in order to suppress or at least modulate immune functions.However, any such therapy attempt has remained without ascertainedtherapeutical success (cf. e.g. Brown, R. H. et al.: Arch. Neurol. 43,383-384, 1986; and e.g. Dalakas, M. C. et al.: Arch. Neurol. 51,861-864, 1994). A disadvantage of such approach is that these forms oftherapy (blind or unaware of specific cell parameters with ALS) are notwell-directed, i.e. they do not block or suppress exclusively those celltypes which are specifically present, or present in significantlyincreased numbers, in ALS patients.

Tests performed by the applicant have shown that the blood of ALSpatients contains mononucleic cells of the immune system which are notfound with any other neurological disease or in healthy persons (ALSspecific immune cells, cf. Table 1). Contrary to the immune cell typesso far observed to be present in increased numbers in ALS patients whichare known as prior art (Schubert, W.; Neurosci. Lett. 198, 29-32, 1995),the cell types discovered according to the invention and listed in Table1 are totally ALS specific. These cell types express receptors forimmunoglobulins (Fcγ receptors), preferably for immunoglobulin G (IgG)of under-class 1 (IgG1) and 3 (IgG3) (in the following referred to asFcγRIII). The increased number, or the ALS specific occurrence, of saidimmune cells is an explanation of the hitherto unexplained reduction ofIgG1 and IgG3 in the serum of patients diagnosed with ALS which wasreported e.g. by Westarp et al. (Westarp, M. E. et al., Neurosci. Lett.173, 124-126, 1994): FcγRIII receptors selectively bind to theseimmunoglobulins, i.e. a multiplication of cells expressing saidreceptors (as discovered in applicant's own studies) will result inincreased binding to IgG1 and IgG3 and thus an IgG1-IgG3 clearance inthis disease. In applicant's own studies, it has been found for thefirst time that these Fcγ receptor-positive cells exhibit a series ofmolecular activation characteristics on the cellular surface and, whencultured, will cause damage to, or even the destruction of, nerve cells.These cells are therefore directly involved in the pathomechanism of ALSand, as a cellular form, for the first time constitute a concrete targetstructure for a well-directed, i.e. specific, therapy with immunoactivesubstances.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is therefore based on the discovery of Fcγreceptor positive, in particular FcγRIII positive, activated cellularforms in the blood stream which are ALS unspecific, or significantlyincreased in number in the case of ALS, and relates to a use ofsubstances for the selective suppression, destruction or selectivefunctional blocking of said cellular forms, or blocking or functionalinactivation of Fcγ receptors, by infusion or injection of definedsubstances as specified in claim 1.

Fcγ receptors have been known for quite some time in immunology. Thereare three different, yet related classes of human Fcγ receptors: FcγRI,FcγRII and FcγRIII. The amino acid sequences of the members of thisreceptor family as well as the genes coding for said receptors areknown: FcγRI (Allen, J. M., Seed B.: Science 243, 378-380, 1989), FcγRII(Ravetch, J. V., Kinet, J.-P.: Annu. Rev. Immunol. 9, 457-492, 1991),FcγRIII (Ravetch, J. V., Perussia, B: J. Exp. Med. 170, 481-497, 1989).Most subtypes of these three receptor classes are anchored in the cellmembrane of certain immune cells. However, all three classes alsocontain soluble receptor proteins, i.e. proteins not anchored in thecell membrane, which are naturally-released by immune cells. Themechanisms are different, however. Soluble FcγRI receptors are generatedby a stop codon in the extracellular domain. Soluble FcγRII receptorsare generated by alternative RNA splicing, and soluble FcγRIII receptorsare generated by proteolytic splitting of the receptor anchored in thecell membrane (cf. summarizing survey in: Jan, G. J. et al.: ImmunologyToday, 14, 215-221, 1993).

Said Fcγ receptors are particularly relevant for a number of importantimmunologic functions. All these functions are based on the fact thatFcγ receptors specifically bind to G immunoglobulins. The binding ofsuch immunoglobulins to Fcγ receptors stimulates or triggers a varietyof different cellular activities: Phagocytosis, endocytosis,antibody-dependent cell-mediated cytotoxicity, the release of soluble,inflammation-promoting factors (mediators) as well as the increase ofantigen presenting mechanisms. G immunoglobulins bind to Fcγ receptorsvia their Fc parts. If G immunoglobulins for example bind to Fcγreceptors anchored on the surface of certain immune cells in thismanner, said immune cells will then be capable of binding veryspecifically to antigen-bearing target cells and destroying them. Thesemechanisms probably play an important pathogenetic part in numerous socalled auto immune diseases. In some cases it has been possible to showthat blocking or functionally inactivating said mechanisms largelyprevents the disease specific destruction of target cells. One examplehereof is a rare form of an auto immune disease in humans, the so calledacute immuno-thrombocytopenic purpura, in which an Fcγ receptor-mediatedcytotoxic activity of the immune system against blood platelets resultsin an acute diminution of said platelets (thrombocytes) in the blood ofaffected patients. In this case, it has been possible to show that theinfusion of soluble Fcγ receptor fragments results in a clear recoveryfrom the symptoms and the cellular signs of the disease (Debr é et al.:Lancet, 342, 945-948, 1993).

In the case of ALS, however, an immunological pathomechanism based onthe activity of Fcγ receptors has not been known so far. Also, a form oftherapy directed at specifically inactivating or blocking such mechanismhas not been developed or conducted.

Now, however, the existence of accordingly specific cellular forms andsuch mechanisms has been confirmed by the results of examinationsconducted by the applicant.

In detail, it has been found

that the blood of ALS patients contains mononucleic cells bearing Fcγreceptors on their cellular surfaces, additionally or simultaneouslybearing a different number of various other receptor proteins whichexhibit an unusual form of cellular surface activation. The surfacereceptors variably co-expressed together with Fcγ receptors, above allFcγRIII receptors, are individually designated in Table 1. Theabbreviations (CD) used therein are in keeping with the internationalnomenclature (cf. Barcley, A. N. et al.; The leucocyte antigen factsbook. Academic press. London. 1993). This special combination of Fcγreceptors (e.g. CD16) with other receptor proteins indicated in Table 1is ALS specific since it could not be detected in healthy persons orwith other neurological control diseases or other immunological,non-neurological diseases;

that these cells, after being isolated from the blood of ALS patients,could be cultivated in cell culture media (the standard protocols onwhich this was based are described in Lindl, T., Bauer, J.: Zell- undGewebekultur, published by G. Fischer Verlag, Stuttgart, 1987),

that said cells, after enrichment and in co-cultivation with nerve cellsand serum of ALS patients, develop a cytotoxic activity resulting in thedestruction of the nerve cells,

that it is possible to block said cytotoxic activity by the addition ofvarious soluble factors:

a) by monoclonal antibodies against Fcγ receptors at a concentration ofbetween 20 and 100 μmol,

b) by soluble Fcγ receptors at a concentration of between 10 and 60μmol,

c) by anti-sense messenger ribonucleic acid species (mRNA) which arecomplementary to Fcγ receptor-specific mRNA sequences (e.g. 20 μmol of ano. 23 anti-sense oligonucleotide, derived from EMBL/gene bank FcγRIIIsequence X16863),

d) by protein V isolated from Gardnerella vaginalis (EP: 0595997) atconcentrations of between 150 to 170 μmol in the simultaneous presenceof serum from ALS patients.

Consequently, the solution according to the invention is the blocking ofthe Fcγ receptor-mediated cytotoxic activity of the ALS-specific Fcγreceptor-positive immune cells, or the blocking or the inactivation ofFcγ receptors, or the destruction thereof, by the administration of theabovementioned substances, so as to prevent the direct or indirectpathological effects of these cells or the Fcγ receptors on themotoneuron system.

Said substances will therefore be applied in the respective therapeuticdosage and after a toleration test in bolus (see the examples).Approximate values are the following concentrations, for example: forFcγR specific antibodies (10 to 1,000 mg/weight kg, once or twice a dayi.v.) , for Fcγreceptor specific anti-sense RNA (10 to 20 mg/kg), forsoluble Fcγ receptors (10 to 1,000 mg/kg), for protein V (10 to 1,000mg/kg).

However, an exact therapeutic dose can only be decided on with respectto the individual case concerned It depends, amongst other things, onthe response of the Fcγ receptor-positive cells to the administration ofthe substances as indicated in claim 1 and on the individual tolerationwhich may be determined by single bolus administrations (see example 1)Such response may e.g. be established by the determination of the numberof such cellular forms in the blood or by in-vitro cytotoxicity assays.If necessary, the dosage and the distribution of the daily dosis mayhave to be modified according to the individual case concerned.

The solution according to the invention has the following advantages:

1. In contrast to all other forms of immunosuppressive orimmunomodulating therapy performed so far which were substantiallyunspecific and affected the entire immune system, the form of therapydisclosed herein constitutes a well-directed form of therapy, i.e.directed at ALS specific forms of immune cells.

2 Based on the knowledge, according to the invention, of the ALSspecific surface characteristics, i.e. the combinatorial receptorpatterns of these cells (see Table 1), it is possible to ascertainbefore each therapy whether these cells are present at all, or, aftertheir isolation and in-vitro testing, to determine whether they exhibitany cytotoxic, in particular neurotoxic, activity.

3 Based on the knowledge, according to the invention, of the cellsurface characteristics of these cells, the therapeutical success cannot only be determined clinically, but also on a cellular level, withthe therapy according to claim 1 in full progress, by the examination ofblood samples for a therapy-related decrease in the number, or even atotal elimination, of the Fcγ receptor-positive cellular forms from theblood stream.

4. Based on the findings of above step 3, the dosage of the administeredsubstances may be adapted accordingly, i.e. increased or decreased,depending on whether or not there is a response of the Fcγreceptor-positive cellular forms.

5. Since the knowledge, according to the invention, of the cell surfacecharacteristics of the ALS specific cellular forms for the first timeconstitutes a disease-specific cell parameter for ALS, it is possible tocorrelate the clinical course of the disease precisely with thisparameter so that, even after a discontinuation of the therapy inaccordance with the inventive muse of Fcγ receptor-specific substances,a potential re-increase of the Fcγ receptor-positive cells will allowexact determination of the time for a further therapy cycle.

EXAMPLES

The solution according to the invention, including the way it works, isdescribed in more detail hereinafter with reference to an embodiment

Example 1

Patient: X1 Diagnosis: ALS, bulbous type Substance: soluble Fcγ receptorpreparations, 50 kDa, from E. coli (fusion proteins from E. coli) Modeof application: intravenous Therapeutical scheme: toleration test with10 to 1,000 mg/kg in bolus i.v., subsequently 150 mg/weight kg daily,over a period of 5 days

TABLE 1 ALS Control CD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 nCD16 + + + + + + + + + + + + + + + + + + CD2 + + + + + − − − − − − − − −− − − − CD3 + + + + + + + + + + + + + + + + + + CD4 + + + + + + +− + + + + + + + − − + CD8 − + − − − − − − − − − − − − − − − − CD56 −− + + + + + + + + + − − − − + + − CD57 + − + − − − − − − − − − − − − − −− CD26 − + − − − − − − − − − − − − − − − − CD38 +− + + + + + + + + + + + + + + + + CD71 − + − − − − − − − − − − − − − − −− HLA−DR + − + + + + + + + + − + + − − − − − HLA−DQ − − − − − − − − − −− − − − − − − − CD11b + − − − − + + − − − − + − + + − − − CD45Ra − + − −− − − − − − − − − − − − − − CD7 + − + − − + − + − − − − + − − − − −CD62L + − + + − + + + + − − + + + − − + + CD36 − + − − − − − − − − − − −− − − − CD19 − − − − − − − − − − − − − − − − CD: “Cluster ofdifferentiation antigens” of mononucleic peripheral blood leucocytesisolated on the cell surface; 1 to 17: different ALS specific cellularforms simultaneously expressing CD16 (FcγRIII) in different combinationswith other CD−surface antigens on the cell surface; n: FcγRIII−positivenormal immune cellular form in a healthy experimentee

What is claimed is:
 1. A method of blocking the cytotoxic activity ofFcγRIII receptor-positive immune cells in a patient with amyotrophiclateral sclerosis, said method comprising the steps of: (a) detectingfor mononucleic cells bearing FcγRIII receptor in a blood sample of saidpatient; and (b) administering to said patient an effective amount ofprotein V, wherein said protein V inhibits the cytotoxic activity ofFcγRIII receptor-positive immune cells in said patient and wherein saidprotein V binds to immunoglobulin G.
 2. The method of claim 1, whereinsaid immunoglobulin G is selected from the group consisting of IgG1andIgG3.
 3. The method of claim 1, wherein said protein V is obtained fromGardnerella vaginalis.
 4. The method of claim 1, wherein saidadministering is by infusion or injection.
 5. The method of claim 1,wherein said protein V binds to IgG1 to block FcγRIII receptor activity.6. The method of claim 1, wherein said protein V binds to IgG3 to blockFcγRIII receptor activity.
 7. The method of claim 1, wherein saidprotein V is administered at a concentration of between 150 and 170μmol.8. The method of claim 1, wherein said protein V is administered to saidpatient in an amount of between 10 to 1,000 mg/weight kg.